Chondrocyte Monolayer Culture

Chondrocytes were dispersed in a suspension of 0.1 x 10⁶ cells/mL in DMEM supplemented with 10% fetal bovine serum (Biowest, France), 10 mM HEPES, 100 U/mL penicillin, 0.1 mg/mL streptomycin, 2 mM glutamine (Lonza, Belgium), 20 μg/mL proline and 50 μg/mL ascorbic acid (Sigma-Aldrich, Belgium) and seeded in 6-well plates at the density of 0.2 x 10⁶ cells/well. Chondrocytes were cultured in monolayer for 5–7 days until 95% confluence. Only primary cultures were used to ensure the stability of the chondrocyte phenotype. Chondrocytes were then cultured 24 h in DMEM supplemented with 1% fetal bovine serum, 10 mM HEPES, 100 U/mL penicillin, 0.1 mg/mL streptomycin, 2 mM glutamine, 20 μg/mL proline and 50 μg/mL ascorbic acid. Afterward, the culture medium was replaced by fresh culture medium containing either 20% (v/v) saline (control), 20% (v/v) Ze14 or 10% (v/v) Ze14 (lower concentration was achieved as follows: 8 parts medium: 1 part saline: 1 part Ze14; corresponding to half of the Ze14 concentration, used only for the transcriptomic study), with or without the addition of human IL-1β 10^–11 M (Roche, Belgium) (Comblain et al., 2016). Cells were incubated for 24 h (transcriptome analysis only) or 72 h (transcriptome and protein analysis). Each culture condition was carried out in triplicates.

For transcriptome analysis, cells were scrapped after 24 h of incubation, three wells were pooled, and ribonucleic acid (RNA) extraction was performed using an RNeasy mini kit according to the instructions of the manufacturer (Qiagen, Netherlands). Cell lysates were stored frozen at −80°C until RNA extraction.

For LDH release assay, conditioned culture media was collected after 24 h of incubation and assayed immediately. Cells were scraped and homogenized in 500 µL of Tris-HCl buffer by ultrasonic dissociation for 20 s at 4°C, to measure total LDH content. Remaining conditioned culture media was stored at −20°C until further analysis.

For protein analyses, conditioned culture media was collected after 24 h and 72 h of incubation and was then stored at −20°C until further analysis. Three wells were used per condition. Cells were trypsinized and homogenized in 500 µL of Tris-HCl buffer by ultrasonic dissociation for 20 s at 4°C to measure total DNA content after 24 h and 72 h of incubation.